Bioinformatics-assisted characterization of the ITS1-58.S-ITS2 segments of nuclear rRNA gene clusters, and its exploitation in molecular diagnostics of European crop-parasitic nematodes of the genus Ditylenchus

In the present study, the determination and computer-assisted characterization of ribosomal DNA (rDNA) sequence data were performed to improve classification and diagnostics of nematodes of the genus Ditylenchus. Dot-plot analyses of the ITS1-5.8S-ITS2 segments revealed the presence of short repeated DNA motifs localized in the ITS1 region of D. destructor, whilst no similar DNA repeats were identified in other members of the genus. Additionally, three-domain secondary structure models were inferred for ITS2 transcripts, providing a valuable resource for complex phylogenetic studies. Finally, alignment of the ITS-rDNA sequence data and subsequent oligonucleotide combinations trials led to the development of taxon-specific amplified regions. Together, this is the first report in which the three most common taxa of crop-parasitic nematodes of the genus Ditylenchus, namely D. dipsaci sensu stricto, Ditylenchus sp. B and D. destructor, have been simultaneously detected using a multiplex polymerase chain reaction (PCR)-based approach. This novel DNA technology is particularly valuable for the precise molecular diagnostics applicable to phytosanitary control and marker-assisted selection (MAS)-based plant breeding.